Scientific basis for the project

Mitochondrial DNA sequences vary between individuals of a given species, although this variation is far less complex than in the nuclear genome. Cats, as humans, can be assigned to different haplotypes or haplogroups based on their mtDNA sequences; all cats of a given haplotype will share the same set of SNPs at key positions in their mtDNA genomes. It is possible to distinguish between mtDNA from different individuals using RFLP analysis by identifying SNPs that introduce or abolish a restriction enzyme digest site.

In this practical, students will be given cat hair from one out of four possible ‘suspects’ and they will have sections of mtDNA sequence from four cats. They will need to map the restriction sites present in the four mtDNA sequences and identify some enzymes for use in RFLP that will allow them to distinguish between the sequences and identify the source of the hair.

Brief summary of each session

Session 1 is a computer-based planning class that requires about 2 hours. The students are given the instructions and are told to plan some experiments to solve the designated problem. They have all of the links and information that they need but we do not tell them which techniques to use. The goal of this activity is to engage them in the experimental design process. Students need to identify SNPs and associated restriction enzymes that would allow them to distinguish between the four potential sources of cat hair based on the mtDNA sequence. We ask them to generate a table of predicted results for each cat and for each restriction enzyme. It is important to check the students’ experimental plans to ensure that they all know what to do.

Session 2 is lab-based and takes 3 hours. Students need to extract the DNA from the cat hairs and then set up a PCR reaction. The PCR takes a couple of hours so session 3 needs to be at least that much later.

Session 3 is also lab-based and requires students to set up restriction digest reactions on their PCR products. They then need to run the digested DNA on an electrophoresis gel and interpret their results.

Learning objectives

This practical is designed to provide students with the opportunity to:

• practise their experimental design and planning skills.
• use online tools and specialist software to investigate DNA sequences, identify restriction enzyme digest sites and construct basic phylogenies.
• develop their molecular biology skills including DNA extraction, PCR, restriction digests and gel electrophoresis.
• interpret the results from gel electrophoresis images.

We are currently perfecting the protocols – please get in touch  with Emma Jones (emma.jones@sheffield.ac.uk) if you are thinking of using this practical. 

If you decide to use this practical, please let us know about it by filling in this short form – it’s not a requirement but we’d love to hear how our ideas are being used!